The present invention relates to a new method of sequencing by oligomer hybridization, DNA/RNA hybrid oligomers applied onto a support and optionally DNA oligomers and/or RNA oligomers being used as hybridization matrix.
In addition to the DNA sequencing known for a long time and effected by the application onto gels, a further method was developed some years ago, namely the sequencing by oligomer hybridization. See, e.g., Drmanac et al., 1989, Genomics 4:114-128; Bains et al., 1988, J. Theor. Biol. 135:303-307. For this sequencing method a complete set of short oligonucleotides and oligomers, respectively, e.g., a set of all conceivable 65.536 octamers, is bound in an ordered grid on a support material, so that the position of each individual oligonucleotide is known. A DNA fragment whose sequence shall be determined is hybridized on such a hybridization matrix and an xe2x80x9coligomer chipxe2x80x9d, respectively, under conditions which only permit a specific duplex formation. As a result, the DNA fragment only binds to the oligomers whose complementary sequence corresponds accurately to a portion, e.g., an octamer when an xe2x80x9coctamer chipxe2x80x9d is used, of its own sequence. By the detection of the binding position of the hybridized DNA fragment, all oligomer sequences present in the fragment are thus determined. On account of the overlap of adjacent oligomer sequences, the continuous sequence of the DNA fragment can be determined by suitable mathematical algorithms. This principle is shown by way of example in FIG. 1 for a DNA fragment having a length of 13 bases, whose sequence shall be determined by octamer hybridization sequencing.
A major problem occurring when this method is practiced consists in that as a function of their sequence, the oligomers have a differing bond strength, i.e., dissociation temperature, but have to bind specifically and in full length to the DNA fragment to be determined under a single set of experimental hybridization temperatures. However, when high stringency is employed, weakly binding oligomers do not bind to the DNA fragment even though their complementary sequence is included therein (false negatives). In the case of low stringency, however, partial regions of oligomers, e.g., only six or seven nucleotides of an octamer, having high bond strength also bind, so that false positives form. Because of the width of the stability differences both artifacts occur under most conditions. For utilizing this sequencing technique it is therefore necessary to bring the bond strength of all oligomers to a level, irrespective of their sequence. By now, this has been done by varying the oligomer concentration. Khrapko et al., 1991, DNA Sequence 1:375-388. However, this is accompanied by the drawback that great technical difficulties occur when the hybridization matrix is produced, since a specific concentration has to be adjusted additionally for every oligomer. A second approach serving for bringing the bond strength of all oligomers to a level is represented by the introduction of base derivatives which result in a change of the stability of the particular base pairing. In connection therewith it is disadvantageous that many differing derivatives have to be used and the specificity of the base pairing is changed by the introduction of the derivatives. A third solution for modulating the bond strength of the oligomers provides for the hybridization in tetraalkylammonium salts which reduce the stability differences existing between G:C and A:T base pairs. Wood et al., 1985, Proc. Natl. Acad. Sci. USA 82:1585-1588. However, this effect is insufficient for short oligomers. Moreover, the use of the highly molar salt solutions impedes the hybridization. Therefore, it was the object of the present invention to bring the bond strength of all oligomers to a level in a sequencing method by means of oligomer hybridization and simultaneously avoid the above described drawbacks.
The present invention relates to a method of sequencing DNA by oligomer hybridization, DNA/RNA hybrid oligomers applied onto a support and optionally DNA oligomers and/or RNA oligomers being used as hybridization matrix.